A variety of enzyme immunoassay techniques are known. These include methods where an enzyme is bound to an antibody or antigen to be detected. Competition between the enzyme labeled species and unknown for the binding partner bound to a solid support is measured. The enzyme remaining in the solution is measured by reaction to substrate.
Homogeneous enzyme immunoassay techniques are described in U.S. Pat. Nos. 4,043,872 (hapten bound to enzyme) and 4,065,354 (hapten bound to lysozyme). Enzyme cofactor labeled ligands are described (Analytical Biochemistry 72, 271 (1976) and ibid 283).
The present invention is particularly distinct in that it involves conjugation of the hapten to be determined to a protein (m.w. 2,000 to 75,000) which is a reversible enzyme inhibitor. Competition between the hapten to be determined and hapten enzyme inhibitor conjugate for antibody to the hapten, in the presence of enzyme, followed by addition of enzyme substrate provides an effective method for hapten analysis.
Colorimetric change is conveniently measured on a bichromatic spectrophotometer of the type described in U.S. Pat. Nos. 3,748,044; 3,831,618; 3,833,304; 3,900,289; 3,817,424; and 3,811,780 (hereinafter referred to as bichromatic analyzer).